Kinetics of carboxymyoglobin and oxymyoglobin studied by picosecond spectroscopy

The dissociation of carboxymyoglobin (MbCO) and oxymyoglobin (MbO2) induced by 530-nm picosecond excitation in the f3 band or the 355-nm 6 band has been measured by monitoring the absorbance changes at 420 and 440 nm corresponding to ligand-bound and ligand-detached species, respectively. We find that MbO2 and MbCO dissociate with very similar rates, which do not reflect the 30-fold difference between the quantum yields of the two reactions. Kinetic data suggest that a short-lived intermediate is formed that is responsible for the low quantum efficiency of the MbO2 dissociation.Ligand detachment in myoglobin and hemoglobin can be initiated by light excitation; therefore the photodissociation ofoxymyoglobin (*MbO2) and carboxymyoglobin (MbCO) is a commonly used method for investigating ligand binding and energy relaxation mechanisms. Several studies have shown that whereas the photodissociation of MbCO occurs with a quantum yield of 1, the photodissociation of MbO2 has a quantum yield of about 0.03. The apparent discrepancy in quantum yields might be elucidated if the rates of MbCO and MbO2 dissociation were known. The methods of Mdssbauer, magnetic resonance, stopped-flow, and nanosecond spectroscopy (1, 2) provide important information and knowledge about the general mechanism of ligand dissociation and cooperativity between hemoglobin subunits. By these methods, it has been possible to study the height and nature ofthe energy barrier(s) ofthe dissociation process and provide support for a dynamic dissociative mechanism (3, 4). However, these methods cannot provide the kinetic data of the initial events in ligand dissociation, which are in the picosecond range and are necessary for the understanding of the dissociation process. To that effect, picosecond spectroscopy has provided some specific information on the rates of ligand dissociation in myoglobin (5) and hemoglobin (6). However, because of the complexity of tetrameric hemoglobin, it is difficult to elucidate the dissociation mechanism and cooperativity among its subunits. We felt that the monomeric nature of myoglobin would remove some of the inherent structural difficulties present for hemoglobin and make the interpretation easier.In previous communications (5, 7) we have described the experimental procedure and preliminary picosecond dissociation data for MbO2 and MbCO, and as a result of these picosecond experiments, it is known that the initial photodissociation of MbCO and MbO2 is a picosecond process. The observed ultrafast relaxation from the electronically excited states is thought to be due, at least in part, to the influence of the metal (8, 9). This interpretation is based on experimental data showing that metal-containing porphyrins exhibit picosecond relaxation rates from their excited ir -iT* levels to the ground state, in contrast to the metal-free porphyrins, which are characterized by relaxation rates three orders ofmagnitude slower. Even with this information, the relaxation pathways have not been established. The experimental ...

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Mechanisms for excited state relaxation and dissociation of oxymyoglobin and carboxymyoglobin.

Reynolds¹,

Rand²,

Rentzepis³

1981

“…We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1)(2)(3)(4)(5)(6), 02 (1,(4)(5)(6), and NO (6,7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.…”

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confidence: 99%

Picosecond fluorescence decay of tryptophans in myoglobin.

Hochstrasser¹,

Negus²

1984

122

The fluorescence decay characteristics of Mb, MbCO, metMb (sperm whale), metMb (yellowfin tuna), and their apo derivatives were determined by using a picosecond streak camera and time-correlated single photon counting. The emission is dominated by tryptophans that transfer their energy to the heme on a subnanosecond time scale. Sperm whale Mb and derivatives have two tryptophans and their decays can be interpreted mainly as two exponentials, one of ca. 20 ps and the other of 130 ps, whereas tuna Mb has one tryptophan and its emission is nonexponential but dominated by one component of 31 ps. These results along with F6rster energy transfer calculations allow us to assign the ca. 30-ps emission to Trp-14 and the 130-ps emission to Mb. The streak camera was modified to determine the decay of the fluorescence anisotropy. In metMb (tuna) the fluorescence anisotropy decays in 100 ps, which is postulated to result from rapid motion of the Trp-14. Because energy transfer was used to gate the anisotropy, the fast motion of Trp-14 is proposed to correspond to only 10% of the equilibrium distribution of molecules.Tryptophans are useful optical probes of protein structure because of their relatively long wavelength absorption compared with polypeptides and other common amino acid residues. A first step in realizing the full potential of such probes is the development of techniques to distinguish the different tryptophans in a protein by means of their optical responses. We have therefore carried out picosecond laser experiments on Mb aimed at characterizing the fluorescent behavior of each of the tryptophans. Previous picosecond laser experiments aimed at detecting rapid conformational changes and relaxation processes in heme proteins have involved studying changes in the heme electronic structure as a result of photodeligation of CO (1-6), 02 (1,(4)(5)(6), and NO (6, 7). A goal of the present research is to pave the way for the study of the transmission of structural changes throughout the protein, and as a first step we have attempted in the present work to chart the picosecond anatomy of the tryptophans of Mb.Pulsed laser techniques are needed to study tryptophan fluorescence in heme proteins as a result of very efficient energy transfer to the heme. The low fluorescence quantum yields for tryptophan in heme proteins (8) suggest that the corresponding lifetimes are in the picosecond range. Thus, to obtain direct information on the lifetimes and motional characteristics of such tryptophans it was necessary to devise experiments by which the fluorescence and its polarization could be measured with picosecond accuracy.Sperm whale (SW) Mb is known to contain the two tryptophan residues, Trp-7 and Trp-14 (9). Thus, it is expected that the emission will be dominated by two decays. The heme is considerably further from Trp-7 than from Trp-14 (20 A compared with 15 A) so that the effects of energy transfer on these excited states are expected to be different. Tuna Mb has an x-ray structure very similar to SW Mb (10) bu...

“…Greene et al (8) found that HbCO responded similarly to 530-and 358-nm pulses. The present study of the 530-nm photolysis of HbCO and HbO2 is aimed at identifying the species initially generated by light and how these species evolve.Photodissociation of liganded hemoglobins is a well-known phenomenon (1) used to study dynamics of ligand binding to deoxyhemoglobin (Hb) (2)(3)(4)(5)(6)(7)(8). The usefulness of the photoprocess for subsequent dynamical studies is restricted at present by a lack of knowledge of the mechanism of the photodissociation.…”

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confidence: 99%

“…Using subpicosecond pulses at 615 nm to excite and probe HbO2 and HbCO, they found induced absorption build-up in less than 0.5 psec for both species, a decay of the HbO2 signal in 2.5 psec, and no decay of the HbCO signal up to 20 psec. Rentzepis and coworkers (6,7) studied HbCO and the myoglobin compounds MbCO and MbO2 by exciting with 6-psec 530-nm pulses and using 440-nm interrogation over the period 0-00 psec. That group reported a significantly longer (11 …”

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confidence: 99%

Geminate recombination of O2 and hemoglobin.

Chernoff¹,

Hochstrasser²,

Steele³

1980

146

The photolysis of HbO2 and HbCO has been studied by measuring transient absorption spectra in the Soret region after excitation with picosecond pulses at 530 nm. Dissociation occurred promptly in both cases, followed (for HbO2) by geminate recombination of ca. 40% of the photodissociated O2 with a lifetime of 200 ± 70 psec (25°C). No to 300 psec. The initial MbCO photoproduct signal at 440 nm was reported to undergo a 15% decay (lifetime r = 125 + 50 psec), but for MbO2 no evidence of decay up to 450 psec was found. Greene et al. (8) conducted a study of HbO2 and HbCO photolysis using 8-psec, 353-nm excitation. They obtained transient absorption spectra in the Soret (400-450 nm) and visible (535-575 nm) regions at 10 psec and 680 psec, finding a persistent, broadened deoxy Hb-like absorption. With their spectra, they observed less than 10% recombination in HbCO over this time interval and less than 20% recombination of photolyzed HbO2. Greene et al. (8) found that HbCO responded similarly to 530-and 358-nm pulses. The present study of the 530-nm photolysis of HbCO and HbO2 is aimed at identifying the species initially generated by light and how these species evolve.Photodissociation of liganded hemoglobins is a well-known phenomenon (1) used to study dynamics of ligand binding to deoxyhemoglobin (Hb) (2)(3)(4)(5)(6)(7)(8). The usefulness of the photoprocess for subsequent dynamical studies is restricted at present by a lack of knowledge of the mechanism of the photodissociation. Of particular interest is the quantum yield for dissociation under steady illumination, which is fairly high (k 0.5) (9, 10) for carboxyhemoglobin (HbCO) but much lower (0 0.05) for the oxy form (HbO2) (9). Two limiting mechanisms can account for the relatively low yield of HbO2 photolysis. First, energy relaxation to nonreactive states may be much faster than the dissociation. Second, although the initial dissociation step in HbO2 may be just as efficient as that in HbCO, some released 02 trapped nearby to the heme may be able to rapidly recombine. These mechanisms are evaluated here by examining the dynamics and identifying the products of the photolysis by picosecond spectroscopy. We have attempted to describe our spectroscopic and dynamical results in terms of recent experimental and theoretical assessments of the excited states of hemoglobins (11-15).The time scale for studying photolysis and subsequent events in heme proteins has in recent years been pressed back to the picosecond regime, starting with the work of Shank et al. (5). Using subpicosecond pulses at 615 nm to excite and probe HbO2 and HbCO, they found induced absorption build-up in less than 0.5 psec for both species, a decay of the HbO2 signal in 2.5 psec, and no decay of the HbCO signal up to 20 psec. Rentzepis and coworkers (6, 7) studied HbCO and the myoglobin compounds MbCO and MbO2 by exciting with 6-psec 530-nm pulses and using 440-nm interrogation over the period 0-00 psec. That group reported a significantly longer (11 psec) predissociation lifetime...